Until recently, it was assumed that cultivation of microorganisms from the environment resulted in the isolation of a good proportion of the microorganisms present. Phylogenetic analysis of rRNA sequences obtained from direct sampling of environments has shown that this is not the case. Giovannoni et al. (1990) Nature 345:60-63; Pace et al. (1996) ASM News 62:463-470; Stahl et al. (1985) Appl. Environ. Microbiol. 49:1379-1384; Suzuki et al. (1997) Appl. Environ. Microbiol. 63:983-989; Ward et al. (1990) Nature 345:63-65. It is now apparent that the microorganisms that can be cultured from any environment using standard techniques probably represent the minority of the total species present in that environment, indicating that a vastly greater diversity of prokaryotes exists than suggested by culturing methods. Pace et al, supra; Stahl (1993) ASM News 59:609-613. The idea that perhaps the vast majority of bacteria in an environment are currently nonculturable has revolutionized thinking in microbiology, and has stimulated new approaches to the study of microbes. Woese et al. (1990) PNAS 87:4576-4579.
For instance, it is estimated that the number of species currently culturable from soil represents 1% or less of the total population. Griffiths et al. (1996) Microbial Ecol. 31:269-280; Torsvik et al. (1996) J. Ind. Microbiol. 17:170-178. DNA-DNA reassociation measurements have been used to determine total genetic diversity in one soil sample. The data indicated that greater than 4000 species might be present. Torsvik et al. (1990) Appl. Environ. Microbiol. 56:782-787. This represented at least 200 times more diversity than was observed by examining culturable bacteria from the same sample. Another study based on methods that did not involve culturing suggested 13,000 species in 100 g soil. Torsvik et al. (1994) p.39-48, In Beyond the Biomass, K. Ritz, J. Dighton and K. E. Giller (eds.), John Wiley and Sons, Chichester. By estimating the total number of cells at 5.times.10.sup.11 per gram of soil, this suggested an average of 5.times.10.sup.7 cells per species assuming even species distribution. Thus even rare species might have fairly large population sizes in the soil. A recent analysis in our labs indicated that novel phyla of Bacteria and Archaea are present in soil. Bintrim et al. (1997) PNAS 94:277-282; Bintrim et al., in press. Of 144 cloned Bacterial 16S rRNA gene sequences examined, 45 had the closest affiliation to members of the phylum Proteobacteria, but of these clones, only 6 had close affiliation to known genera (Pseudomonas, Hafnia, Azospirillum). The clones were distributed across the entire Domain, and none were identical to any previously known sequence. Moreover, these studies revealed entirely new lineages of microbes in soil, both from the Domain Archaea and the Domain Bacteria. This indicates the enormous diversity of noncultured microorganisms from soil.